Chemotaxis plays a critical role in many physiological processes such as innate immunity, lymphocyte homing, angiogenesis, embryogenesis, neurogenesis, and wound healing. Knowledge of its basic mechanisms would provide therapeutic targets for multiple disease states such as asthma, multiple sclerosis, arthritis, metastasis, and atherosclerosis. In chemotaxing cells specific phosphoinositides (PIs), recognized by PH-domains, accumulate at the cell's leading edge and play a key role in pseudopodia formation. Recent evidence shows that PI 3-kinases (PI3Ks) and PI 3-phosphatase (PTEN) bind to the membrane at the front and back of the cell, respectively, and thereby control PI localization. These discoveries are leading to a novel paradigm for chemotaxis and suggest further experiments proposed here. First, to elucidate PI circuits and their control of directional pseudopodia formation, the regulation of PI3K and PTEN will be studied in Dictyostelium discoideum cells lacking these enzymes, expressing constitutive versions, and with combinations of these deficiencies. Plans are to correlate this data with measurements of actin polymerization, other physiological responses, and chemotactic behavior. Second, the PI3K and PTEN holoenzymes will be purified and the genes for anticipated regulatory subunits identified. Studies are designed to map regions of the catalytic and regulatory subunits that control chemoattractant-mediated enzyme activation and membrane binding and, for PTEN, to determine the basis of a novel chemoattractant-induced electrophoretic mobility shift. Third, a series of approaches are designed to identify the membrane binding sites for PI3K and PTEN. Strategies include characterization of a new in vitro reconstitution assay where stimuli generate the membrane sites that bind exogenous PI3K, identification of membrane proteins phosphorylated during receptor and G-protein stimulation, and the use of fragments of the catalytic or regulatory subunits of PI3K and PTEN as specific probes to tag the membrane binding sites. Fourth, genetic selections to find negative regulators or enhancers of activation and discover links of PIs to downstream responses are planned. These include suppressor screens of the aggregation deficient phenotypes of PI3K and PTEN and direct microscopic observation of defects in chemoattractant regulation of membrane binding of the enzymes as well as the selection of mutants that circumvent the inhibitory effects of persistent chemoattract stimulation.
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